Tissue and organ transplantation is a worldwide therapeutic approach for end-stage organ failure (Nasseri et al., “Tissue Engineering: An Evolving 21st-Century Science to Provide Biological Replacement for Reconstruction and Transplantation,” Surgery 130:781-784 (2001)). Currently, over 100,000 people are in need of an organ transplant in the United States alone. Each day, 17-20 patients die while waiting for donor organs. These statistics highlight the worsening problem facing transplant patients—the demand for tissues and organs far outweighs the available supply. The field of tissue engineering offers great potential for reducing the number of patient deaths associated with this shortage of organs. By developing methods for repairing or replacing diseased or injured tissues and organs, tissue engineering aims to provide an alternative supply of tissues and organs to balance supply and demand (Langer et al., “Tissue Engineering,” Science 260:920-926 (1993)). Before such a goal can be fully realized, tissue engineers need to successfully reconstitute viable tissues and organs in vitro, a task that depends on the delivery of essential nutrients and oxygen to all cells within the tissue to uphold their metabolic processes. Existing delivery methods include the passive diffusion of oxygen and nutrients through the tissue and host-dependent vascularization of the tissue after implantation (Nomi et al., “Principals of Neovascularization for Tissue Engineering,” Molecular Aspects of Medicine 23:463-483 (2002); Lokmic et al., “Engineering the Microcirculation,” Tissue Engineering 14(1):87-103 (2008); Tremblay et al., “Inosculation of Tissue-Engineered Capillaries with the Host's Vasculature in a Reconstructed Skin Transplanted on Mice,” American Journal of Transplantation 5:1002-1010 (2005)). These methods are limited to tissues with less than a few millimeters in thickness and have therefore, only been successfully used for the development of skin replacements (Folkman et al., “Self-Regulation of Growth in Three Dimensions,” The Journal of Experimental Medicine 138:745-753 (1973); Mooney et al., “Growing New Organs,” Scientific American 280(4):60-65 (1999); Nomi et al., “Principals of Neovascularization for Tissue Engineering,” Molecular Aspects of Medicine 23:463-483 (2002); Tremblay et al., “Inosculation of Tissue-Engineered Capillaries with the Host's Vasculature in a Reconstructed Skin Transplanted on Mice,” American Journal of Transplantation 5:1002-1010 (2005); Griffith et al., “Diffusion Limits of an in Vitro Thick Prevascularized Tissue,” Tissue Engineering 11(1-2):257-266 (2005)). As such, the engineering of larger, more complex, three-dimensional (3D) tissues and organs requires the in vitro development of a vascular system throughout the construct to adequately provide oxygen and nutrients to all areas of the tissue (Griffith et al., “Tissue Engineering-Current Challenges and Expanding Opportunities,” Science 295:1009-1014 (2002); Nerem R. M., “Tissue Engineering: The Hope, the Hype, and the Future,” Tissue Engineering 12:1143-1150 (2006); Jain et al., “Engineering Vascularized Tissue,” Nature Biotechnology 23(7):821-823 (2005); Levenburg et al., “Engineering Vascularized Skeletal Muscle Tissue,” Nature Biotechnology 23(7):879-884 (2005)).
Successful induction of neovessel network formation in tissue constructs depends on the stimulation of endothelial cell functions critical to angiogenesis. Endothelial cell survival, growth, migration and differentiation are influenced by the spatial distribution of endothelial cells and the organization of surrounding extracellular matrix (“ECM”) (Korff et al., “Integration of Endothelial Cells in Multicellular Spheroids Prevents Apoptosis and Induces Differentiation,” The Journal of Cell Biology 143(5):1341-1352 (1998); Ino et al., “Application of Magnetic Force-Based Cell Patterning for Controlling Cell-Cell Interactions in Angiogenesis,” Biotechnology and Bioengineering 102(3):882-890 (2009); Vailhe et al., “In Vitro Models of Vasculogenesis and Angiogenesis,” Laboratory Investigation 81(4):439-452 (2001); Nehls et al., “The Configuration of Fibrin Clots Determines Capillary Morphogenesis and Endothelial Cell Migration,” Microvascular Research 51:347-364 (1996); Vailhe et al., “In Vitro Angiogenesis is Modulated by the Mechanical Properties of Fibrin Gels and is Related to Alpha-v Beta-3 Integrin Localization,” In Vitro Cell. Dev. Biol.-Animal 33:763-773 (1997); Ingber et al., “Mechanochemical Switching between Growth and Differentiation During Fibroblast Growth Factor-Stimulated Angiogenesis In Vitro: Role of Extracellular Matrix,” The Journal of Cell Biology 109:317-330 (1989); Stephanou et al., “The Rigidity in fibrin Gels as a Contributing Factor to the Dynamics of In Vitro Vascular Cord Formation,” Microvascular Research 73:182-190 (2007); Sieminski et al., “The Relative Magnitudes of Endothelial Force Generation and Matrix Stiffness Modulate Capillary Morphogenesis In Vitro,” Experimental Cell Research 297:574-584 (2004; and Montesano et al., “In Vitro Rapid Organization of Endothelial Cells into Capillary-Like Networks is Promoted by Collagen Matrices,” The Journal of Cell Biology 97:1648-1652 (1983), which are hereby incorporated by reference in their entirety). As such, control over both endothelial cell and ECM protein organization within 3D tissue constructs will affect endothelial cell functions essential to angiogenesis.
Technologies currently in development to organize cells and proteins into complex patterns can be divided into two general categories. In the first approach, micropatterning of cell-adhesive contacts using extracellular matrix proteins coated onto microfabricated stamps by photolithography or microcontact printing is used to direct cell adhesion into pre-designed patterns. In the second approach, a force is applied to cells to direct cell movement to a desired location. The applied force can be optical, magnetic, electrokinetic, or fluidic ((Lin et al., “Dielectrophoresis Based-Cell Patterning for Tissue Engineering,” Biotechnol J 1:949-57 (2006)). The ability of acoustic radiation forces associated with ultrasound standing wave fields to control the spatial distribution of cells and extracellular matrix proteins in a three-dimensional tissue model has not previously been investigated.
When an ultrasonic pressure wave is incident on an acoustic reflector, the reflected wave interferes with the incident wave resulting in the development of an ultrasound standing wave field (USWF). An USWF is characterized by areas of maximum pressure, known as pressure antinodes, and areas of zero pressure, known as pressure nodes. Exposure of particle or cell suspensions to an USWF can result in the alignment of particles or cells into bands that are perpendicular to the direction of sound propagation and that are spaced at half-wavelength intervals (Coakley et al., “Cell Manipulation in Ultrasonic Standing Wave Fields,” J Chem Tech Biotechnol 44:43-62 (1989); Gould et al., “The Effects of Acoustic Forces on Small Particles in Suspension,” In: Finite amplitude wave effects in fluids: Proceedings of the 1973 Symposium, Guildford: IPC Science and Technology Press Ltd, Bjorno L, ed. pp. 252-7 (1974); and Whitworth et al., “Particle Column Formation in a Stationary Ultrasonic Field,” J Acoust Soc Am 91:79-85 (1992)). A primary acoustic radiation force, (Frad), generated along the direction of sound propagation in the USWF, is largely responsible for this movement. Frad is defined as
                              F          rad                =                              (                                                            -                  π                                ⁢                                                                  ⁢                                  P                  o                  2                                ⁢                V                ⁢                                                                  ⁢                                  β                  o                                                            2                ⁢                λ                                      )                    *          ϕ          *                      sin            ⁡                          (                                                4                  ⁢                  π                  ⁢                                                                          ⁢                  z                                λ                            )                                                          (                  Equation          ⁢                                          ⁢          1                )            where Po is the USWF peak pressure amplitude, V is the spherical particle volume, λ is the wavelength of the sound field, z is the perpendicular distance on axis from pressure nodal planes, and φ is an acoustic contrast factor given by
                    ϕ        =                                                            5                ⁢                                  ρ                  p                                            -                              2                ⁢                                  ρ                  o                                                                                    2                ⁢                                  ρ                  p                                            +                              ρ                o                                              -                                    β              p                                      β              o                                                          (                  Equation          ⁢                                          ⁢          2                )            where ρp and βp are the density and compressibility of the particles or cells, and ρo and βo are the density and compressibility of the suspending medium, respectively (Gol'dberg Z A, “Radiation Forces Acting on a Particle in a Sound Field,” In: High Intensity Ultrasonic Fields, New York: Plenum Press, Rozenberg L D, ed., pp. 109-17 (1971); Gor'kov L P, “On the Forces Acting on a Small Particle in an Acoustical Field in an Ideal Fluid,” Sov Phys Dokl 6:773-5 (1962); and Gould et al., “The Effects of Acoustic Forces on Small Particles in Suspension,” In: Finite amplitude wave effects in fluids: Proceedings of the 1973 Symposium, Guildford: IPC Science and Technology Press Ltd, Bjorno L, ed. pp. 252-7 (1974). The forces generating the banded pattern exist only during application of the USWF. Therefore, to maintain the USWF-induced banded distribution, suspending mediums have been used that undergo a phase conversion from a liquid to a solid state during USWF exposure (Gherardini et al., “A Study of the Spatial Organisation of Microbial Cells in a Gel Matrix Subjected to Treatment With Ultrasound Standing Waves,” Bioseparation 10:153-62 (2002); Gherardini et al., “A New Immobilisation Method to Arrange Particles in a Gel Matrix by Ultrasound Standing Waves,” Ultrasound Med Biol 31:261-72 (2005); Saito et al., “Fabrication of a Polymer Composite With Periodic Structure by the Use of Ultrasonic Waves,” J Appl Phys 83:3490-4 (1998); and Saito et al.,” “Composite Materials With Ultrasonically Induced Layer or Lattice Structure,” Jpn J Appl Phys 38:3028-31 (1999)). In this way, the banded pattern is retained after removal of the sound field.
The present invention is directed to overcoming these and other deficiencies in the art.